Topical microbicides, a female-controlled strategy to prevent HIV, are crucial in stemming the pandemic. An optimal microbicide should protect against infection without disrupting the mucosal environment or its mediators of host defense. This project focuses on the preclinical evaluation of combination microbicides with the goal of identifying safe and effective prevention strategies. Combinations that target at least two steps in the HIV life cycle will be prioritized, thus providing protection against circulating resistant viruses and reducing the risk that the drugs will select for resistant variants. This will be accomplished through the dual activities of a single antiretroviral drug (ARV), a pyrimidinedione, which is a potent reverse transcriptase inhibitor and also blocks HIV entry, or by combining two different ARV microbicides. The biological synergy between HIV and HSV will be addressed by evaluating the possibility of delivering acyclovir in combination with the ARV microbicides to provide local sustained suppression of HSV replication. The rationale for focusing on HSV suppression reflects the pre-existing high prevalence of HSV-2 (60-90%) in the developing world and the overwhelming biological and epidemiological evidence demonstrating that subclinical HSV recurrences, which are quite common, increase the risk for HIV acquisition. Critical gaps in microbicide development are the lack of biomarkers predictive of safety and efficacy. This project will address these gaps by developing and expanding novel assays, which focus on the interactions between microbicides and the vaginal environment, using primary cells, explant cultures, and a dual chamber culture system and murine models. The proposed safety models focus on the impact of sustained drug delivery on epithelial integrity and genital tract mucosal immunity. The biological significance of any observed changes will be assessed by examining changes in the ability of HIV to traverse the epithelial barrier in the dual culture model and changes in the susceptibility to genital herpes in the mouse. Results obtained from these studies will provide crucial information for the advancement of novel safe and effective combination microbicides.